The airway epithelial mucosal barrier is the heart of a powerful innate immune system that maintains the underlying epithelial surface. At the broadest level, the major scientific goal of this proposal is to elucidate the organization of the airway gel layer, which is formed by very large glycoproteins termed mucins (MUC5B and MUC5AC) coupled with other protective biomolecules through labile interactions, and assess the role of these interactions in airway mucus integrity as an essential pre-requisite for therapeutic intervention. Our hypothesis is that normal airway mucus is highly structured, containing an excess of 100 proteins that form distinct protein complexes many of which are centered around these mucins. These complexes constitute a discrete secretory entity we call the 'mucin interactome'. We propose that gel properties emerge from a dynamic interplay between the mucins and proteins and that these structures engender specific rheological properties of mucus that tune it to removal by cough and flow clearance. This proposal will change the paradigm that mucins are the only molecules that give most of the rheological properties to mucus and will ultimately lead to a greater understanding of the role of these interactions as an innate defense of the lung. In testing these hypotheses, using a broad range of biochemical, molecular and cellular biology methods, we propose: 1) To determine our target panel of proteins that show evidence of specific mucin binding; 2) To determine the domains required for the mucin-protein interactions and 3) To assess the effects of mucin-protein interactions on the surface and bulk rheological properties of the mucus. If the goals of this proposal are achieved, they will increase our knowledge and understanding of the relationship between protein composition and the function of airway secretions. Such knowledge is an essential pre-requisite for informed therapeutic intervention.